Supplementary Figures

SHUGOSHINs and PATRONUS protect meiotic centromere cohesion in Arabidopsis thaliana

Linda Zamariola1, Nico De Storme1, Katrijn Vannerum1, Klaas Vandepoele2,3, Susan J. Armstrong4, F. Chris H. Franklin4, Danny Geelen1

GENE PROPOSED PROTEIN FUNCTION (TAIR) NASC line

At4g35620 Cyclin-B2;2 (CYCB2;2) SALK_067657C

At2g16270 Unknown protein SALK_037235

At1g18370 HIK (HINKEL); microtubule motor SALK_056766C

At5g48310 Similar to unknown protein Arabidopsis thaliana: At4g24610.1 SALK_134597C

At1g76310 Core cell cycle genes;CYCB2;4 (CYCLIN B2;4); cyclin-dependent protein kinase regulator SALK_073438C

At2g44190 Similar to unknown protein Arabidopsis thaliana: At3g60000.2 SALK_047950C

At3g11520 Encodes a B-type mitotic cyclin SALK_026335C

At1g18040 Cyclin-dependent kinase D;3 SALK_151657C

At3g51280 Similar to male sterility MS5 family protein Arabidopsis thaliana: At5g48850.1 SALK_046426C

At5g15510 Similar to unknown protein Arabidopsis thaliana: At3g01015.1 SALK_096426C

At3g55660 Encodes a member of KPP-like gene family, homolog of KPP (kinase partner protein) gene in tomato. SALK_114559C

At4g17000 Unknown protein SALK_131195C

At3g14190 Similar to unknown protein Arabidopsis thaliana: At5g12360.1 SALK_070337; SALK_035661

At1g77720 Encodes a predicted protein kinase based on sequence similarity SALK_121668

At4g14330 Similar to AtK1 (Arabidopsis thaliana KINESIN 1): At4g21270 SALK_145633

At5g01910 Unknown protein SALK_123731

At1g03780 Homolog of vertebrate TPX2 SALK_064340; SAIL_732_CO2

At5g48720 Encodes XRI1 (X-ray induced 1) SALK_056851; SAIL_1162_AO2

At3g23890 Encodes a topoisomerase II that is highly expressed in young seedlings SALK_069748

At1g34770 Unknown protein GK-534A03.01

AT3G44050 Microtubule motor activity, ATP binding WiscDsLox4G07

Table S1List of 21 genes obtained from the integrative comparative genomic analysis, their putative protein function, and the T-DNA lines screened for their pollen phenotype, ordered from NASC.

Figure S1Schematic representation of the intron (black lines) and exon (black rectangulars) structure of the PANS1, PANS2, AtSGO1 and AtSGO2 genes and location of the T-DNA insertions (triangles). The white rectangulars correspond to the 5’ and 3’ UTR regions. Below PANS1 gene are indicated the primers used for gene expression analysis.

pans2-2

pans1-1

pans1-2PANS fw PANS re

Atsgo1-2Atsgo1-1

Atsgo2-1

At3g14190-PANS1

At5g12360-PANS2

At3g10440-AtSGO1

At5g04320-AtSGO2

Bra_001538

Bra_021538

Tha_lv10021564m

Cru_v10014729m

PANS1_AT3G14190

Bra_008894

Cru_v10003502m

Tha_lv10014740m

PANS2_AT5G12360

Cpa_evm.model.supercon

Stub_PGSC0003DMP400035465

Sly_07g055110.2.1

Vvi_06G10710

Ptr_09G07440

Phvul_008G200100.1

Gma_02G44960

Gma_14G03810

Sly_03g025160.2.1

Egr_K00289.1

Osa_RSS1

Zma_ LOC100275076

Gra_011G137900.1

Tca_1EG019923t1

Ccl_10030388m

Csi_orange1.1g039037m

Stub_PGSC0003DMP400017927

Figure S2Phylogenetic tree showing PANS1 and PANS2 conservation among Eudicots. The tree was computed with MEGA 4.0 using neighbor joining method (NJ) with the Poisson correction and 500 bootstrap samples. Internal nodes were labeled with boostrap support values. Rice RSS1 and its maize homolog are also shown in the tree. Bra: Brassica rapa; Tha: Thellungiella halophila; Cru: Capsella rubella; Cpa: Carica papaya; Stub: Solanum tuberosum; Sly: Solanum lycopersicum; Vvi: Vitis vinifera; Ptr: Populus trichocarpa; Phvul: Phaseoulus vulgaris; Gma: Glycine max; Csi: Citrus sinensis; Ccl: Citrus clementina; Egr: Eucalyptus grandis; Tca: Theobroma cacao; Gra: Gossipium raimondii ; Osa: Oryza sativa; Zma: Zea mays.

Figure S3Alignment of PANS1 and PANS2 homologous proteins and identification of three conserved motifs. Colors are reported according to the aminoacid properties, as displayed in the results from the MEME analysis.

DEN box

D box

Unknown motif

Figure S4Male meiotic spread of a) Col-0 wt and b) pans1-1 at prometaphase II, showing abnormalities in the organelle band configuration of pans1-1. Scale bar = 10 µm.

a b

Figure S5Comparison of Col-0 wt, Atsgo1-2 and Atsgo1-2 Atsgo2-1 phenotypes. Pollen distribution of a) Col-0 wt, b) Atsgo1-2, c) Atsgo1-2(-/-)Atsgo2-1(+/-) and d) Atsgo1-2(-/-)Atsgo2-1(-/-) analysed with Multisizer 3, Coulter Counter. d) Tetrads analysis of Col-0 wt, Atsgo2-1, Atsgo1-1, Atsgo1-2, Atsgo1-2(-/-)Atsgo2-1(-/-) . e) Table representing the level of fertility, evaluated counting the number of seeds per silique, and the level of female fertility, evaluated by fertilizing mutant pistils with wt pollen, of Col-0 wt, Atsgo2-1, Atsgo1-1, Atsgo1-2, Atsgo1-2(-/-)Atsgo2-1(+/-) and Atsgo1-2(-/-)Atsgo2-1(-/-) . n.a. = not analyzed.

Genotype Number of seeds per silique

Number of seeds per silique obtained from crosses with Col-0 wt pollen on mutant pistils

Col-0 wt 45,9±8,7 (n=36)Atsgo1-1(-/-) SK35523 24,3±4,4 (n=35) 24,8±6,8 (n=11)Atsgo1-2(-/-) SK2556 21,6±6,4 (n=47) 8,5±6,1 (n=23)Atsgo2-1(-/-) 48,4±8,2 (n=42) n.a.Atsgo1-2(-/-) Atsgo2(+/-) 3,8±1,5 (n=40) 3,3±2,1 (n=33)Atsgo1-2(-/-) Atsgo2(-/-) 3,1±2,0 (n=40) 2,9±2,6 (n=28)e

Col-0 wt

Atsgo1-2

Atsgo1-2(-/-)

Atsgo2-1(-/-)

a b

Atsgo1-2(-/-)

Atsgo2-1(+/-)

c d

f

Figure S6FISH on Col-0 wt (a-c), pans1-1 (d-f) and Atsgo1-2 Atsgo2-1 (g-i) meiocytes. Pachytene (a,a’, d,d’, g,g’), diakinesis (b,b’, e,e’, h,h’), metaphase I (c,c’, f,f’, i,i’). Scale bar = 10 µm.

a a’

b b’

c c’

d d’

e e’

f f’

g g’

h h’

i i’

Col-0 wt pans1-1 Atsgo1-2 Atsgo2-1